The goal of this project has been to establish the role of domain structure and determine the structural requirements for the biological activity of follistatin, a multidomain protein that binds and bioneutralizes activin in pituitary, bone and a wide range of other tissues and physiological systems. Our investigations to date of the 288-residue follistatin molecule have concentrated on functional effects of chemical modifications and mutagenesis on activin binding, as well as on association with cell-surface heparan-sulfate proteoglycans that promote follistatin's tissue localization. These have shown that the 63-residue N-terminal domain plus the first two of the three distinctive 10-cysteine "follistatin" (FS) domains are required for activin binding, and that hydrophobic residues are particularly important both to activin interaction and maintenance of domain conformation. Under this renewal, we will use complementary approaches of crystallography and mutational analysis to examine interdomain relationships and the organization of activin-binding components to provide a functional model of the follistatin molecule. Under Aim 1 we will determine the 3-dimensional structure of the purified follistatin-288 molecule by crystallization and x-ray diffraction analysis. Structures will be done on full-length follistatin and (if needed to enhance resolution) two-domain segments, purified from 293F-cell suspension cultures in serum-free medium. In Aim 2 we will prepare follistatin complexed with activin for crystallography to directly identify contact sites and document changes undergone by key determinants during complex formation. Mutational analyses of the activin ligand under Aim 3 will complement and functionally validate the structures developed from crystallographic analysis of the activin-follistatin complex. Identification of functionally important hydrophobic surface residues will test our hypothesis that follistatin acts through competition with known hydrophobic sites in the activin receptor. These investigations will clarify the mechanism of the widely-postulated local regulation of activin by follistatin, enhance understanding of the activity of other potent binding proteins (such as noggin and chordin) characteristic of the TGF-beta family and expand our insight into the role of follistatin-domain structures in general among the many other extracellular proteins in which they can be found.